In scanning microscopy, especially in confocal microscopy, a specimen to be imaged is not illuminated in its entirety, but instead is scanned point by point in a raster motion by means of a scanning beam, usually emitted from a laser light source. For scanning of an individual specimen point, a scanning time interval during which the scanning beam illuminates the respective specimen point is provided. The radiation emitted from that specimen point, for example fluorescent light triggered by the scanning beam, is then sensed by a detector and converted into an image point signal. Lastly, a raster image signal is assembled from the image point signals generated for the individual specimen points, and on the basis of that signal a raster image depicting the specimen in its entirety can be presented.
The individual image point signals reproduce the brightness or intensity of the radiation that is emitted from an individual specimen point during the pertinent scanning time interval. In order to sense the radiation intensity as precisely as possible, it is conceivable for the intensity of the radiation emitted from the specimen point currently being scanned to be sensed repeatedly during the respective scanning time interval, in order then to determine an intensity mean value from the sensed intensities.
The resulting raster image signal, which reproduces the imaged specimen in its entirety, is assembled in this case from individual mean value image point signals that each represent the intensity or brightness averaged over the respective scanning time interval.
The raster images obtained in the manner described above contain exclusively the specimen brightness as image information. In order to allow, for example, dynamic processes such as diffusion to be analyzed, it would be desirable if the raster images that are generated could supply image information that goes beyond brightness.
Additional image information of this kind can be obtained, for example, in fluorescence correlation spectroscopy. With this method, however, the image information is generally sensed only for a single image point, i.e. an evaluatable raster image cannot be generated therewith. This is possible with so-called raster image correlation spectroscopy (RICS), which works with autocorrelation of the image information. With this method, however, the raster motion must occur at a speed that is in the same range as the speed with which the dynamic processes to be analyzed, for example diffusion, take place. Furthermore, evaluation with this method is particularly complex if the raster motion is not linear. Mention may also be made, in the sector of correlation spectroscopy, of an analytical method referred to in the literature as “number and brightness” (abbreviated “N&B”). This method requires multiple images in order to allow conclusions as to dynamic processes based on the fluctuation at a specific image position.